The aim of the research project is a detailed understanding of the chemical reactivity of the flavin coenzymes, and how their chemical versatility is channeled and controlled in biological systems. The methods of approach will be the study of specific enzymes representative of most of the main classes of flavoproteins and studies of model systems with free flavins. Considerable use will be made of steady state kinetics coupled with rapid reaction kinetics studies, and of the technique of replacing the natural coenzymes of fiavoproteins with synthetic flavins, in order to test possible mechanisms, and to probe the nature of the environment immediately around the protein-bound flavin. This technique is not only particularly important in providing information about the active site region in proteins where structures are not available from x-ray crystallography, but in addition offers a powerful way for investigating dynamic aspects of protein structure in the case of enzymes where the crystal structure is known. We are also making extensive use of molecular genetics techniques in the elucidation of chemical reaction mechanisms. We have cloned and expressed the genes for L-lactate monooxygenase and Old Yellow Enzyme, and through our collaborators have access to genetically modified forms of L-lactate oxidase, alkyl hydroperoxide reductase and theioredoxin reductases. For all of these enzymes, three-dimensional structures are already available, or the structural determination is in progress.